Receptor-mediated endocytosis in rat liver: purification and enzymic characterization of low density organelles involved in uptake of galactose-exposing proteins.

Abstract

Rat liver organelles involved in receptor-mediated endocytosis were labeled with a conjugate of galactosylated BSA to horseradish peroxidase ([3H]galBSA-HRP), injected 10 min before sacrifice. These organelles were recovered at low density (1.11-1.13 g/ml) in sucrose gradients. Upon incubation of such low density fractions in 3,3''-diaminobenzidine (DAB) and H2O2 and equilibration in a 2nd sucrose gradient, galBSA-HRP-containing particles selectively shifted towards heavier densities, resulting in up to 250- to 300-fold purification with respect to the homogenate. The most purified preparations, wherein DAB-stained structures represented .apprx. 85% of the total volume of particles, contained only trace activities of enzymes usually regarded as markers for other subcellular entities. These minor activities could reflect either contamination or true enzyme association to the ligand-containing structures. Considering the latter hypothesis, at most 1.0% of alkaline phosphodiesterase I and 2.6% of 5''-nucleotidase (markers for plasma membrane), 3.6% of N-acetyl-.beta.-glcosaminidase (lysosomes), and 6.0% of galactosyltransferase (Golgi complex) from the homogenate would be associated with the whole population of ligand-containing organelles. After DAB cytochemistry on liver fixed 10 min after galBSA-HRP injection, ligand-containing structures accounted for 0.78-0.89% of the fractional volume of the hepatocytes and displayed a membrane area of 2100 cm2/cm3, compared with 6700 cm2/cm3 for the pericellular membrane. The hypothesis that these ligand-containing organelles are structurally distinct from plasma membrane, lysosomes and Golgi complex is supported.